Anneke P. Bech

Association of anti-PLA₂R antibodies with outcomes after immunosuppressive therapy in idiopathic membranous nephropathy.

BACKGROUND The optimal timing and duration of immunosuppressive therapy for idiopathic membranous nephropathy (iMN) have been debated. This study aimed to evaluate whether measuring the antibody against the phospholipase A2 receptor (PLA2R-ab) at start and end of therapy predicts long-term outcome and therefore may inform this debate. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This observational study included all consecutive high-risk patients with progressive iMN observed from 1997 to 2005 and treated with oral cyclophosphamide (CP) or mycophenolate mofetil (MMF) in combination with corticosteroids for 12 months. Patients were prospectively followed, and outcome was ascertained up to 5 years after completion of immunosuppressive therapy. Serum samples were collected before and after completion of therapy. PLA2R antibodies were determined retrospectively in stored samples using ELISA. RESULTS In total, 48 patients (37 men) were included. The median age was 55 years (range, 34-75), and the median serum creatinine level was 1.60 mg/dl (range, 0.98-3.37 mg/dl). Twenty-two patients received MMF and 26 received CP. At baseline, PLA2R-abs were present in 34 patients (71%). Baseline characteristics and outcome did not significantly differ between patients negative or positive for PLA2R-ab. In PLA2R-ab-positive patients, treatment resulted in a rapid decrease of antibodies: median anti-PLA2R-ab, 428 U/ml (range, 41-16,260 U/ml) at baseline and 24 U/ml (range, 0-505 U/ml) after 2 months. The PLA2R-ab levels at baseline did not predict initial response, but antibody status at end of therapy predicted long-term outcome: After 5 years, 14 of 24 (58%) antibody-negative patients were in persistent remission compared with 0 of 9 (0%) antibody-positive patients (P=0.003). CONCLUSIONS These data suggest that in PLA2R-ab-positive patients, measuring PLA2R-abs at the end of therapy predicts the subsequent course.

Hepatocyte Nuclear Factor 1β–Associated Kidney Disease: More than Renal Cysts and Diabetes

Hepatocyte nuclear factor 1β (HNF1β)-associated disease is a recently recognized clinical entity with a variable multisystem phenotype. Early reports described an association between HNF1B mutations and maturity-onset diabetes of the young. These patients often presented with renal cysts and renal function decline that preceded the diabetes, hence it was initially referred to as renal cysts and diabetes syndrome. However, it is now evident that many more symptoms occur, and diabetes and renal cysts are not always present. The multisystem phenotype is probably attributable to functional promiscuity of the HNF1β transcription factor, involved in the development of the kidney, urogenital tract, pancreas, liver, brain, and parathyroid gland. Nephrologists might diagnose HNF1β-associated kidney disease in patients referred with a suspected diagnosis of autosomal dominant polycystic kidney disease, medullary cystic kidney disease, diabetic nephropathy, or CKD of unknown cause. Associated renal or extrarenal symptoms should alert the nephrologist to HNF1β-associated kidney disease. A considerable proportion of these patients display hypomagnesemia, which sometimes mimics Gitelman syndrome. Other signs include early onset diabetes, gout and hyperparathyroidism, elevated liver enzymes, and congenital anomalies of the urogenital tract. Because many cases of this disease are probably undiagnosed, this review emphasizes the clinical manifestations of HNF1β-associated disease for the nephrologist.

Statistical significance versus clinical relevance

Abstract In March this year, the American Statistical Association (ASA) posted a statement on the correct use of P‐values, in response to a growing concern that the P‐value is commonly misused and misinterpreted. We aim to translate these warnings given by the ASA into a language more easily understood by clinicians and researchers without a deep background in statistics. Moreover, we intend to illustrate the limitations of P‐values, even when used and interpreted correctly, and bring more attention to the clinical relevance of study findings using two recently reported studies as examples. We argue that P‐values are often misinterpreted. A common mistake is saying that P < 0.05 means that the null hypothesis is false, and P ≥0.05 means that the null hypothesis is true. The correct interpretation of a P‐value of 0.05 is that if the null hypothesis were indeed true, a similar or more extreme result would occur 5% of the times upon repeating the study in a similar sample. In other words, the P‐value informs about the likelihood of the data given the null hypothesis and not the other way around. A possible alternative related to the P‐value is the confidence interval (CI). It provides more information on the magnitude of an effect and the imprecision with which that effect was estimated. However, there is no magic bullet to replace P‐values and stop erroneous interpretation of scientific results. Scientists and readers alike should make themselves familiar with the correct, nuanced interpretation of statistical tests, P‐values and CIs.

A Novel Hypokalemic-Alkalotic Salt-Losing Tubulopathy in Patients with CLDN10 Mutations.

Mice lacking distal tubular expression of CLDN10, the gene encoding the tight junction protein Claudin-10, show enhanced paracellular magnesium and calcium permeability and reduced sodium permeability in the thick ascending limb (TAL), leading to a urine concentrating defect. However, the function of renal Claudin-10 in humans remains undetermined. We identified and characterized CLDN10 mutations in two patients with a hypokalemic-alkalotic salt-losing nephropathy. The first patient was diagnosed with Bartter syndrome (BS) >30 years ago. At re-evaluation, we observed hypocalciuria and hypercalcemia, suggesting Gitelman syndrome (GS). However, serum magnesium was in the upper normal to hypermagnesemic range, thiazide responsiveness was not blunted, and genetic analyses did not show mutations in genes associated with GS or BS. Whole-exome sequencing revealed compound heterozygous CLDN10 sequence variants [c.446C>G (p.Pro149Arg) and c.465-1G>A (p.Glu157_Tyr192del)]. The patient had reduced urinary concentrating ability, with a preserved aquaporin-2 response to desmopressin and an intact response to furosemide. These findings were not in line with any other known salt-losing nephropathy. Subsequently, we identified a second unrelated patient showing a similar phenotype, in whom we detected compound heterozygous CLDN10 sequence variants [c.446C>G (p.(Pro149Arg) and c.217G>A (p.Asp73Asn)]. Cell surface biotinylation and immunofluorescence experiments in cells expressing the encoded mutants showed that only one mutation caused significant differences in Claudin-10 membrane localization and tight junction strand formation, indicating that these alterations do not fully explain the phenotype. These data suggest that pathogenic CLDN10 mutations affect TAL paracellular ion transport and cause a novel tight junction disease characterized by a non-BS, non-GS autosomal recessive hypokalemic-alkalotic salt-losing phenotype.

Effects of sildenafil, metformin, and simvastatin on ADH-independent urine concentration in healthy volunteers

Nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by resistance of the kidney to the action of antidiuretic hormone (ADH), resulting in a decrease in the capacity of the kidney to concentrate the urine. NDI can be inherited or acquired due to, for example, chronic lithium therapy. Current treatment options are limited to attempts to lower urine output by a low‐solute diet and the use of diuretics or anti‐inflammatory drugs. These measures are only partially effective. Recent reports suggested that sildenafil, metformin, and simvastatin might improve ADH‐independent urine concentration. If confirmed, this would provide interesting additional therapeutic options for patients with NDI. We, therefore, tested the effect of these drugs on ADH‐independent urine concentrating capacity in healthy volunteers. We included 36 healthy volunteers who received sildenafil 20 mg thrice daily, metformin 500 mg thrice daily or simvastatin 40 mg once daily during 1 week. At baseline and at the end of treatment, a water loading test was performed. No significant increase in lowest urine osmolality was seen after the use of metformin or sildenafil (P = 0.66 and P = 0.09 respectively). Lowest urine osmolality increased modestly but significantly after the use of simvastatin (70 mOsm/kg to 85 mOsm/kg, P = 0.05). Our data suggest that only simvastatin has an effect on urine osmolality in healthy volunteers. Validation studies are needed and, most importantly, these drugs should be tested in patients with NDI.

Thiazide Responsiveness Testing in Patients With Renal Magnesium Wasting and Correlation With Genetic Analysis: A Diagnostic Test Study

To the Editor: Electrolyte disorders such as hypomagnesemia and hypokalemia can result from excessive urinary losses. Rarely, this is due to loss-of-function mutations in genes such as those encoding tubular transporters or ion channels. In Gitelman syndrome (GS), mutant NCCT (encoded by SLC12A3) in the distal convoluted tubule leads to hypokalemia, metabolic alkalosis, hypomagnesemia, and hypocalciuria. Because thiazide diuretics block NCCT, thiazide challenge can probe NCCT function in patients thought to have GS. This thiazide test differentiates well between the salt-losing nephropathies GS and Bartter syndrome (BS). However, the differential diagnosis of GS includes more disorders characterized by renal magnesium wasting (Item S1). The clinical value of the thiazide test in these renal magnesium-wasting disorders is not known. We performed a diagnostic study in patients with renal magnesium wasting, using thiazide responsiveness as the index test and genetic analysis as the reference standard. All patients with renal magnesium wasting (FeMg . 2.7% with hypomagnesemia) and clinical suspicion of GS who had a thiazide test at our institute between 2010 and 2014 were included (detailed methods in Item S1). Thiazide tests were performed as described by Colussi et al. A normal response was considered DFeCl . 2.3%. Genetic testing was done by Sanger sequencing and multiplex ligation–dependent probe amplification. Patient characteristics at the time of presentation to our clinic are given in Table 1. Most patients were receiving magnesium and/or potassium salts at referral. As shown in the thiazide test findings in Fig 1, the 3 patients with an SLC12A3 mutation had a blunted response to hydrochlorothiazide administration, whereas the patient with BS with heterozygous mutations in CLCNKB and KCNJ1 had a normal to increased response. The 5 patients with HNF1B mutations had responses ranging from normal to completely unresponsive. The patient with an FXYD2 mutation showed a clearly blunted response. Thus, a blunted response to thiazide diuretics is not specific for GS because it can also occur in patients with a GS-like syndrome (including magnesiuria and hypocalciuria) who have other underlying genetic causes of renal magnesium wasting (eg, HNF1B or FXYD2 mutations). This could indicate that NCCT function is also affected in these patients. This is remarkable because mutations in FXYD2 are thought to cause isolated renal hypomagnesemia and mutations in HNF1B cause a multisystem phenotype often consisting of renal cysts, diabetes, and reduced kidney function, but its tubulopathy is characterized by an isolated magnesium reabsorption defect as well. The underlying mechanism of presumed NCCT dysfunction in these diseases is unresolved. It could be due to the magnesiuria itself, but both we and others have shown that patients with BS who have magnesiuria respond normally to thiazide diuretics. Therefore, an interaction between tubular transporters or a tubular development defect seems more likely.

Reference values of renal tubular function tests are dependent on age and kidney function

Electrolyte disorders due to tubular disorders are rare, and knowledge about validated clinical diagnostic tools such as tubular function tests is sparse. Reference values for tubular function tests are based on studies with small sample size in young healthy volunteers. Patients with tubular disorders, however, frequently are older and can have a compromised renal function. We therefore evaluated four tubular function tests in individuals with different ages and renal function. We performed furosemide, thiazide, furosemide‐fludrocortisone, and desmopressin tests in healthy individuals aged 18–50 years, healthy individuals aged more than 50 years and individuals with compromised renal function. For each tubular function test we included 10 individuals per group. The responses in young healthy individuals were in line with previously reported values in literature. The maximal increase in fractional chloride excretion after furosemide was below the lower limit of young healthy individuals in 5/10 older subjects and in 2/10 patients with compromised renal function. The maximal increase in fractional chloride excretion after thiazide was below the lower limit of young healthy individuals in 6/10 older subjects and in 7/10 patients with compromised renal function. Median maximal urine osmolality after desmopressin was 1002 mosmol/kg H2O in young healthy individuals, 820 mosmol/kg H2O in older subjects and 624 mosmol/kg H2O in patients with compromised renal function. Reference values for tubular function tests obtained in young healthy adults thus cannot simply be extrapolated to older patients or patients with compromised kidney function. Larger validation studies are needed to define true reference values in these patient categories.